The major objective of this proposal will be to evaluate membrane perturbations occurring during myocardial ischemia and reflow that are hypothesized to be involved in the development of irreversible myocardial injury. Mechanisms of membrane injury that will be evaluated include: 1) oxygen-derived free radical induced lipid peroxidative damage and 2) phospholipase activation resulting in membrane phospholipid degradation and free fatty acid release. The contribution of free radical or phospholipase mediated events in initiating electrolyte alterations will be measured by analytical electron microscopy. The relationship between these mechanisms and their relative contribution to the injury process will be analyzed. Interventions to inhibit each of these events as well as combined interventions will be evaluated. Isolated perfused heart preparations made ischemic and reperfused will be used for these studies. Indices of damage to be evaluated will include measurements of lipid peroxidation products, phospholipid degradation, free fatty acids, electrolyte alterations, myocardial function, adenine nucleotides, enzyme loss, and structural integrity. Superoxide dismutase, catalase, alpha-tocopherol, the chromane compound and phytyl chains of tocopherol, and lodoxamide will be used to examine the contribution of free radical mediated damage. Mepacrine, a phospholipase inhibitor, will be used to evaluate the contribution of phospholipases in myocardial injury. In hearts not subjected to ischemia and reflow, exogenous phospholipases or melittin will be evaluated and compared to ischemic injury. Free radical generating systems will be used to evaluate the production of peroxidative products. Generating systems will also be applied to cultured myocytes to examine altered ionic calcium flux using the fluorescent calcium indicator, fura-2. Treated and nontreated groups will be statistically compared and correlations analyzed for indices of injury, particularly for overlap between peroxidative measurements and free fatty acids, and the various treatments. The proposed studies will provide extensive information concerning the significance of membrane phospholipids in the maintenance of cell survival, alterations in electrolytes and the ability of pharmacological interventions to protect against specific aspects of the damage. Interactions between events capable of altering membrane integrity will be more fully elucidated and the relative importance of these phenomenon in myocardial injury established.